Method for crosslinking hyaluronic acid; method for preparing an injectable hydrogel; hydrogel obtained; use of the obtained hydrogel

Abstract

The present invention relates to a method for crosslinking hyaluronic acid, a method for preparing an injectable hydrogel, the hydrogel thus obtained and its use.

Claims

1. A method for crosslinking hyaluronic acid, or one of its salts, comprising at least the steps of: a) preparing a first aqueous phase of partly crosslinked hyaluronic acid comprising: i) adding hyaluronic acid, or one of its salts, in an aqueous solution to obtain a hyaluronic acid concentration C1, ii) adding an amount Q1 of crosslinking agent, and iii) achieving partial crosslinking of the hyaluronic acid by controlling the temperature T1 and the duration t1 of the crosslinking reaction, wherein the prepared first aqueous phase of partially crosslinked hyaluronic acid is not converted into particles; b) preparing at least one second aqueous phase of partly crosslinked hyaluronic acid comprising: i) adding hyaluronic acid, or one of its salts, in an aqueous solution to obtain a hyaluronic acid concentration C2, ii) adding an amount Q2 of crosslinking agent, and iii) achieving partial crosslinking of the hyaluronic acid by controlling the temperature T2 and the duration t2 of the crosslinking reaction, wherein the prepared at least one second aqueous phase of partially crosslinked hyaluronic acid is not converted into particles; c) adding the at least one second partly crosslinked hyaluronic acid phase into the first aqueous phase, and then producing a mixture of the at least two phases by optionally adding an additional amount Qm of crosslinking agent; and d) continuing the crosslinking of the mixture by controlling the crosslinking temperature Tm and the duration tm of the crosslinking reaction, wherein the crosslinking during steps a), b), c) and d) does not involve the addition of crosslinked hyaluronic acid particles or one of its salts.

2. The method of claim 1, wherein the second aqueous phase of partly crosslinked hyaluronic acid has a partial degree of crosslinking greater than that of the first aqueous phase of partly crosslinked hyaluronic acid and/or in that the hyaluronic acid concentration C2 is greater than or equal to the concentration C1.

3. The method of claim 1, wherein the amount of crosslinking agent Q2 is greater than or equal to the amount Q1 and/or in that the crosslinking temperature T2 is greater than or equal to the temperature T1.

4. The method of claim 1, wherein the crosslinking duration t2 is greater than or equal to the duration t1 and/or in that the respective masses of the first and of the second partly crosslinked hyaluronic acid aqueous phases in the mixture are different.

5. The method of claim 1, wherein the crosslinking of the mixture is stopped by dilution with an aqueous solution and/or by removing the unreacted crosslinking agent by carrying out purification achieved by dialysis.

6. The method of claim 1, wherein the crosslinking reactions are carried out by the action of one or more polyfunctional crosslinking agents selected from the group consisting of bi-functional epoxies, poly-functional epoxies, divinylsulfone, carbodiimides and formaldehyde.

7. The method of claim 6, wherein the crosslinking agent is 1,4-butanedioldiglycidylether (BDDE).

8. The method of claim 1, wherein the salts of hyaluronic acid are selected from the group consisting of sodium, calcium, zinc and potassium.

9. The method of claim 1, wherein the hyaluronic acid, or one of its salts, in step a), or in step b), or in steps a) and b) exhibits a molecular mass of between 0.1 and 4 million Daltons.

10. The method of claim 1, wherein the hyaluronic acid, or one of its salts, in step a), or in step b), or in steps a) and b) is a derivative of hyaluronic acid obtained by modifying the hyaluronic acid via a chemical route, or via any other route.

11. A method for preparing an injectable hydrogel, comprising at least the following successive steps: a) crosslinking of hyaluronic acid, or of one of its salts, according to claim 1, b) purifying the crosslinked hyaluronic acid by means of an iso-osmolar solution and having a suitable pH, c) homogenizing the crosslinked hyaluronic acid, and optionally adding one or more other biocompatible polymers and/or one or more active substances, d) optionally degassing and/or freeze drying, e) conditioning in a syringe, in a flask, or in any other hermetic container, and f) sterilizing.

12. A hydrogel obtained according to the method of claim 11, comprising at least crosslinked hyaluronic acid or one of its salts, wherein the crosslinked hyaluronic acid is in a mixture of at least two crosslinked hyaluronic acid phases having different degrees of crosslinking, the phases being bound to each other through covalent bonds, wherein the total concentration of hyaluronic acid or of one of its salts is comprised between 0.01 and 50 mg/ml.

13. The hydrogel of claim 12, further comprising one or more active substances, having pharmacological action or not, selected from the group consisting of antioxidants, anti-inflammatories, antiseptics, antibacterial agents, antifungal agents, anticancer agents, proteins, hormones, local anesthetics, biological entities, and combinations thereof.

14. The hydrogel of claim 12, comprising lidocaine and/or one or more mineral substances.

15. A kit comprising the hydrogel of claim 12, wherein the hydrogel has been conditioned in sterile syringes.

16. The hydrogel of claim 12 for use in esthetic or therapeutic applications or for formulating an intradermally or subcutaneously implantable composition for improving the quality of the skin or filling the wrinkles or restoring volumes of the face or of the body, for the formulation of a cosmetic or cosmeceutical composition, for the formulation of a topical or intra-ocular composition, for applications in ophthalmology, for the formulation of a topical or implantable composition for applications in dentistry, for the formulation of an intra-articular composition for applications in orthopedia and rheumatology, for the formulation of an implantable composition in urology, for applications in the treatment of incontinence, for the formulation of a topical or implantable composition used in medicine or general surgery within the scope of treating fibrosis or for improving healing of wounds, for the formulation of a topical or implantable pharmaceutical composition allowing delayed and/or controlled release of active substances for various medical applications.

17. The method of claim 1, wherein steps a) i) and/or b) i) comprise adding other biocompatible polymers, and wherein the crosslinking during steps a), b), c) and d) does not involve the addition of crosslinked biocompatible polymers.

18. The method of claim 1, wherein the crosslinking temperature Tm is greater than the temperature T2.

19. The method of claim 1, wherein the crosslinking duration t2 is greater than or equal to the duration t1 and/or in that the respective masses of the first and of the second partly crosslinked hyaluronic acid aqueous phases in the mixture are equal.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. For gel 2: the partly crosslinked phases C and D.

(2) FIG. 2. For gel 2: the partly crosslinked phases C and D.

(3) FIG. 3. For gel 2: the crosslinked product obtained from the partly crosslinked phases C and D.

(4) FIG. 4. For gel X: the particles Xp.

(5) FIG. 5. For gel X: the crosslinked product obtained by co-crosslinking of particles Xp.

(6) FIG. 6. For gel Y: the particles Yp.

(7) FIG. 7. For gel Y: the crosslinked product obtained by co-crosslinking of particles Yp.

EXAMPLES

(8) The invention will now be illustrated in a non-limiting way by the following examples.

(9) Sodium hyaluronate, sodium carboxymethylcellulose, lidocaine hydrochloride, phospho-calcium hydroxyapatite and the whole of the other compounds used in the examples have a high degree of purity.

(10) The injectability of the prepared gels was determined by measuring the required force (in Newtons) for ejecting the gel contained in a glass syringe of 1 ml (BD Hypak SCF, 1 ml long RF-PRTC) through a given needle at a rate of 12.5 mm/min.

(11) The rheological properties of the gels were measured at 25° C. by means of an AR2000 rheometer (TA Instruments) using a plane-plane geometry of 40 mm and a gap of 1000 μm.

Example 1

Preparation of a Gel 1 According to the Invention

(12) a) Preparation of a Partly Crosslinked Phase A

(13) 4.70 g of sodium hyaluronate (NaHA) fibers with a molecular mass of about equal to 3.0 MDa and having a humidity level of 13.8% were weighed, to which 45.75 g of an aqueous 1 wt. % NaOH solution were added.

(14) The hydration of the fibers took 1 h 20 min, with regular manual homogenization with a spatula.

(15) 1.41 g of a solution of 1,4-butanediol diglycidylether (BDDE) diluted to 1:5 in a 1 wt. % NaOH solution were added to the reaction medium, followed by manual homogenization for 15 min.

(16) The gel thus obtained was divided into three equal masses to prepare the following three fractions: Fraction 1 was introduced into a thermostatic bath at 30° C. for 1 h and was then diluted with a phosphate buffer solution containing HCl to obtain a concentration of NaHA of 20 mg/ml and a neutral pH (=phase A′). Phase A′ was mechanically homogenized for 10 min (=gel A′). Fraction 2 was introduced into a thermostatic bath at 50° C. for 2 h and was then diluted with a phosphate buffer solution containing HCl to obtain a concentration of NaHA of 20 mg/ml and a neutral pH (=phase A″). Phase A″ was mechanically homogenized for 10 min (=gel A″). Fraction 3 was introduced into a thermostatic bath at 30° C. for 1 h. Let A be the obtained partly crosslinked phase.
b) Preparation of a Partly Crosslinked Phase B

(17) 5.25 g of sodium hyaluronate (NaHA) fibers with a molecular mass of about equal to 3.0 MDa and having a humidity level of 13.8% were weighed, to which 45.75 g of an aqueous 1 wt. % NaOH solution were added.

(18) The hydration of the fibers took 1 h 20 min, with regular manual homogenization with a spatula.

(19) 2.73 g of a solution of 1,4-butanediol diglycidylether (BDDE) diluted to 1:5 in a in a 1 wt. % NaOH solution were added to the reaction mixture, followed by 15 min of manual homogenization with a spatula.

(20) The gel thus obtained was divided into three equal masses to prepare the following three fractions: Fraction 1 was introduced into a thermostatic bath at 30° C. for 1 h and was then diluted with a phosphate buffer solution containing HCl to obtain a concentration of NaHA of 20 mg/ml and a neutral pH (=phase B′). Phase B′ was mechanically homogenized for 10 min (=gel B′). Fraction 2 was introduced into a thermostatic bath at 50° C. for 2 h and was then diluted with a phosphate buffer solution containing HCl to obtain a concentration of NaHA of 20 mg/ml and a neutral pH (=phase B″). Phase B″ was mechanically homogenized for 10 min (=gel B″). Fraction 3 was introduced into a thermostatic bath at 30° C. for 1 h. Let B be the obtained partly crosslinked phase.
c) Combining the Partly Crosslinked Phases A and B and Continuing the Crosslinking

(21) 9.0 g of the partly crosslinked phase A were added into 13.0 g of the partly crosslinked phase B and subjected to mechanical mixing for 15 min at room temperature before immersion into a thermostatic bath at 50° C. for 2 h.

(22) The crosslinked product was neutralized to pH=7.0 in a phosphate buffer solution containing HCl, in which it was allowed to swell for 24 h and then it was dialyzed for 24 h with a phosphate buffer solution in order to remove unreacted BDDE.

(23) After mechanical homogenization for 10 min, a gel 1 having a sodium hyaluronate concentration of 20 mg/ml was obtained.

Example 2

Characterization of the Gel 1 of Example 1

(24) The gels 1, A′, A″, B′ and B″ were introduced into glass syringes of 1 ml and the forces required for extruding the gels through a 27G½ needle at a rate of 12.5 mm/min are indicated in the table below.

(25) TABLE-US-00001 Tested gel Ejection force (N) 1 23.3 A′ 6.1 B′ 7.0 A″ 6.7 B″ 25.1

(26) It is seen that: the crosslinking reactions are partial for phases A and B. Indeed, the ejection forces of A″ and B″ are greater than that of A′ and B′, respectively, demonstrating that the gels A″ and B″ are more crosslinked than A′ and B′, and the gel 1 according to the invention has an ejection force lower than that of the B″ and, thus, has a better injectability than the gel that has not been subjected to the crosslinking method according to the invention.

(27) The rheological viscoelasticity properties G′ and G″ of gel 1 at 1.0 Hz are also measured.

(28) TABLE-US-00002 Tested gel G′ (1.0 Hz) in Pa G″ (1.0 Hz) in Pa 1 96 0.40

Example 3

Preparation of a Gel 2 According to the Invention

(29) a) Preparation of a Partly Crosslinked Phase C

(30) 4.2 g of sodium hyaluronate (NaHA) powder with a molecular mass of about equal to 2.0 MDa and having a humidity level of 6.3% were weighed, to which 0.3 g of sodium carboxymethylcellulose (42 mPa.Math.s, 2% in water, at 20° C.) with a humidity level of 2.0%, and 33.6 g of an aqueous 1 wt. % NaOH solution were added.

(31) The hydration of the powder took 1 h 20 min, with regular manual homogenization with a spatula.

(32) Let H be the obtained hydrated phase.

(33) 1.80 g of a 1,4-butanediol diglycidylether (BDDE) solution diluted to 1:5 in a 1 wt. % NaOH solution were added to 27.0 g of phase H, then a manual homogenization with a spatula was carried out for 15 min, followed by introducing the reaction mixture into a thermostatic bath at 50° C. for 1 h 30 min.

(34) b) Preparation of a Partly Crosslinked Phase D

(35) 0.10 g of a 1,4-butanediol diglycidylether (BDDE) solution diluted to 1:5 in a 1 wt. % NaOH solution were added to 3.0 g of phase H obtained above, then a manual homogenization with a spatula was carried out for 15 min, followed by introducing the reaction mixture into a thermostatic bath at 50° C. for 2 h.

(36) c) Combining the Partly Crosslinked Phases C and D and Continuing the Crosslinking

(37) The partly crosslinked phase D was added to the partly crosslinked phase C and was subjected to mechanical mixing for 15 min at room temperature before immersion into a thermostatic bath at 50° C. for 2 h 30 min.

(38) The crosslinked product was neutralized to pH=7.0 in a phosphate buffer solution containing HCl, in which it was allowed to swell for 24 h and then it was dialyzed for 24 h with a phosphate buffer solution in order to remove unreacted BDDE.

(39) After mechanical homogenization for 10 min, a gel 2 having a sodium hyaluronate concentration of 20 mg/ml was obtained.

Example 4

Preparation of an Injectable Formulation 1 According to the Invention

(40) In 25% of the mass of gel 2 obtained in Example 3, 0.3 wt. % of lidocaine hydrochloride powder was added and mechanical homogenization was carried out for 15 min.

(41) The thus obtained gel was introduced into 1 ml glass syringes, and then the syringes were sterilized in an autoclave at 121° C. for 20 min.

Example 5

Preparation of an Injectable Formulation 2 According to the Invention

(42) In 25% of the gel mass 2 obtained in Example 3, 30 wt. % of a phospho-calcium hydroxyapatite powder with a particle size of between 30 and 50 μm was added and mechanical homogenization was carried out for 15 min.

(43) The thus obtained gel was introduced into 1 ml glass syringes, and these syringes were then sterilized in an autoclave at 121° C. for 20 min.

Example 6

Characterization of the Injectable Formulation 1 of Example 4

(44) The sterile formulation obtained in Example 4 was found to be easily injectable through a thin 27G½ needle; an injection force of 21.3 N was measured at a rate of 12.5 mm/min.

(45) The pH and the osmolarity of this formulation are physiological: pH=6.9 Osmolarity=290 mOsm/kg H.sub.2O

(46) The rheological characterizations resulted in an elastic modulus G′ of 74 Pa and a Tan δ of 0.25 at a frequency of 1.0 Hz.

(47) It is important to note that these rheological characteristics are compatible with injection into the dermis or subcutaneously.

(48) In two 10 ml flasks F1 and F2 containing 8 ml of purified water, were introduced: 1 ml of Restylane® (Galderma Q-Med, Uppsala, Sweden) gel into F1; Restylane® is a product based on crosslinked hyaluronic acid at 20 mg/ml known for more than 10 years in esthetic medicine, and 1 ml of the injectable formulation 1 into F2.

(49) F1 and F2 were manually stirred for 5 seconds. After a further 10 seconds, it was seen that the Restylane® product is completely dissociated in water (suspension of crosslinked hyaluronic particles in water), which is not the case for the injectable formulation 1 which was always in the form of a gel in water. The cohesivity level of the injectable formulation 1 is therefore much higher than that of the Restylane® product.

Example 7

Comparison of the Production of a Gel According to the Invention with the Prior Art (US 2010/0028435)

(50) a) Preparation of a Gel 2 According to the Invention

(51) See Example 3.

(52) b) Preparation of a Prior Art Gel X According to Example 4 of US Application 2010/0028435

(53) 3.50 g of sodium hyaluronate (NaHA) powder with a molecular mass of about equal to 2.0 MDa and having a humidity level of 6.3% were weighed, to which 24.0 g of an aqueous 1 wt. % NaOH solution were added.

(54) The hydration of the powder took 1 h, followed by manual homogenization for 10 min with a spatula.

(55) 700 μl of BDDE were added to the reaction medium, followed by manual homogenization for 10 min with a spatula before immersion into a thermostatic bath at 50° C. for 2 h.

(56) The crosslinked product was neutralized to pH=7.0 in a phosphate buffer solution containing HCl, in which it was allowed to swell for 24 h (end volume=92 ml).

(57) The gel thus obtained was grinded into particles by serial passage of the same through a grid of 250 μm.

(58) Let Xp be the obtained particles.

(59) 3.50 g of sodium hyaluronate (NaHA) powder with a molecular mass of about equal to 2.0 MDa and having a humidity level of 6.3% were weighed, to which 30.5 g of an aqueous 1 wt. % NaOH solution were added.

(60) The hydration of the powder took 1 h followed by a manual homogenization of 10 min with a spatula.

(61) 263 μl of BDDE were added to the reaction medium, followed by a manual homogenization for 10 min with a spatula before immersion into a thermostatic bath at 50° C. for 2 h.

(62) Let Xg be the obtained crosslinked product.

(63) 58 g of particles Xp and 24 g of an aqueous 1 wt. % NaOH solution were added to the crosslinked product Xg, followed by homogenization with a spatula for 10 min and continuation of the crosslinking reaction for 6 h at 25° C.

(64) The crosslinked product was neutralized to pH=7.0 in a phosphate buffer solution containing HCl, in which it was allowed to swell for 24 h, and then it was dialyzed for 24 h with a phosphate buffer solution in order to remove unreacted BDDE.

(65) After mechanical homogenization for 30 min, a gel X was obtained.

(66) c) Comparison of the Production Processes of Gel 2 and Gel X

(67) The gel 2 and the gel Y were filled into glass syringes of 1 ml and extruded through needles of 27G½ (6 tests for each gel/manual injection by an operator). The gel 2 and the gel X were easily injectable through the needle, however, it is important to note that the gel 2 according to the invention was found to have a much better homogeneity within the syringe. In fact, during the injections, the operator carrying out the test observed many irregularities with the X gel (not the case with gel 2 of the invention) with regard to the force that must be applied to the syringe plunger; irregularities characterized by the operator as “pushs”. On the other hand, among the six syringes tested for gel X, two syringes could not be completely discharged because the needle was completely blocked/obstructed by the gel (not the case with gel 2 according to the invention).

(68) As described above, the prior art teaches that the conversion of a crosslinked gel into particles enables to decrease the ejection force of the product and, thus, to enhance its injectability through a needle (the strongly crosslinked hydrogel is grinded into particles of small size that are more likely to pass through the needle hole). Surprisingly, the gel 2 of the invention, which was not subjected to any grinding step of partially crosslinked phases (one of which is highly crosslinked and the other being less crosslinked), but has a crosslinked phase obtained from combining and crosslinking of the said two partly crosslinked phases, was also found to be readily injectable and also more homogeneous. The complex “multistructured” (and without co-crosslinked) structure according to the invention makes it therefore possible to address the problem of injectability described in the prior art without having to grind the product to convert it into particles.

(69) Toluidine blue staining tests were performed on gel samples taken during the process of preparing gel 2 and gel X in order to evaluate if the structure of the gel is particulate or not. For this purpose, about 0.1 g of gel to be tested were disposed on a glass slide and five drops of an aqueous solution of toluidine blue (0.1 g toluidine blue per 100 g of deionized water) were added, and then a second glass slide was placed on the first glass slide. The result was then viewed under a microscope at a magnification of 40× and a picture was taken.

(70) The following color tests were performed: For gel 2: for the partly crosslinked phases C and D (see FIGS. 1 and 2) and for the crosslinked product obtained from the partly crosslinked phases C and D (see FIG. 3), after neutralization to a neutral pH for three samples; For gel X: for the particles Xp (see FIG. 4) and for the crosslinked product obtained by co-crosslinking of particles Xp (see FIG. 5), after neutralization to a neutral pH for two samples.

(71) In contrast to FIG. 4 (and also FIG. 5), in which NaHA crosslinked particles are clearly visible, the gel structure of FIGS. 1 to 3 relating to gel 2 according to the invention is not particulate. The non-conversion of the partly crosslinked phases during the crosslinking method of the invention allows one to obtain a hydrogel which does not contain particles of co-crosslinked NaHA.

(72) This represents a significant advantage over the prior art products, and especially over the products described in US 2010/0028435 and also in EP 2 011 816. Indeed, as described in the literature and also in the applications US 2010/0028435 and EP 2 011 816, the crosslinked particles generate side effects and complications in the short term as well as in the long term (in EP 2 011 816, crosslinked particles are specifically described as generating more or less strong reactions against foreign matters). In the present invention, the absence of co-crosslinked particles thus represents a major improvement over the inventions disclosed in US 2010/0028435 and EP 2 011 816 with respect to a better safety profile of the product while remaining easily injectable.

(73) On the other hand, it is also important to note that US 2010/0028435 and EP 2 011 816 claim to limit migration of highly crosslinked particles within the body due to the co-crosslinking of these particles with a weakly crosslinked NaHA matrix. This is true in the short term, but the migration of highly crosslinked particles becomes possible again in the longer term, when the matrix of cross-linked weakly crosslinked NaHA has been resorbed. This latter point further corroborates the improved safety profile of the injectable hydrogel according to the invention, where the problem of migration of co-crosslinked particles in the long term does not arise (the hydrogel according to the invention does not contain co-crosslinked particles).

(74) Another major advantage is the better integration (=bio-integration/bio-implantation) into tissues of the product according to the invention compared with the products of co-crosslinked particles known from US 2010/0028435 and also from EP 2 011 816. Indeed, as described in various scientific publications (e.g., Tran C. et al., In vivo bio-integration of three HA fillers in human skin: a histological study, Dermatology 2014, 228:4754; or Micheels, P. et al., Superficial dermal injection of HA soft tissue fillers: comparative ultrasound study, Dermatol. Surg. 2012, 38:1162-1169), the injection of products based on particulate, crosslinked NaHA into the dermis (e.g., of the products disclosed in US 2010/0028435 and EP 2 011 816) are distributed significantly more heterogeneous as compared to products based on non-particulate, crosslinked NaHA (as in case of the hydrogel according to the invention).

(75) It is also important to note that the method of preparing a hydrogel according to the invention requires a significantly shorter time for manufacturing a product compared to the methods described in US 2010/0028435 and EP 2 011 816. A comparison of the manufacturing time for the gel 2 of the invention (approximately 55 h) and gel X according to US 2010/0028435 (approximately 85 h) may illustrates this point. This provides a clear economic advantage and also minimizes the risk of bacterial contamination of the gel during manufacturing, which increases with duration of the manufacturing process.

Example 8

Comparison of the Production of a Gel According to the Invention with the Prior Art (EP 2 011 816)

(76) a) Preparation of a Gel 2 According to the Invention

(77) See Example 3.

(78) b) Preparation of a Prior Art Gel Y According to Example 1 of EP Application 2 011 816

(79) 1.00 g of sodium hyaluronate (NaHA) powder with a molecular mass of about equal to 2.0 MDa and having a humidity level of 6.3% were weighed, to which 6.2 g of an aqueous 1 wt. % NaOH solution were added.

(80) The hydration of the powder took 1 h 20 min, with regular manual homogenization with a spatula.

(81) 0.15 g of BDDE were added to the reaction medium, followed by manual homogenization for 10 min with a spatula before immersion into a thermostatic bath at 50° C. for 2 h 30 min.

(82) The crosslinked product was introduced into deionized water for 24 h, wherein three successive baths, each for 8 h, were used, in order to remove unreacted BDDE.

(83) The gel thus obtained was grinded into particles by serial passage of the same through a grid of 250 μm and the particles were left to drain for 2 h.

(84) Let Yp be the obtained particles.

(85) 5.0 g of particles Yp were added to 0.8 g of sodium hyaluronate (NaHA) powder with a molecular mass of about equal to 2.0 MDa and having a humidity level of 6.3%, to which 2.0 g of an aqueous 1 wt. % NaOH solution were added.

(86) The mixture was manually homogenized for 30 min before addition of 0.03 g BDDE and then again homogenized for 30 min before immersion of the reaction mixture into a thermostatic bath at 50° C. for 2 h 30 min.

(87) Let Yg be the obtained crosslinked product.

(88) The crosslinked product Yg was neutralized to pH=7.0 in a phosphate buffer solution containing HCl, in which it was allowed to swell for 24 h, and then it was dialyzed for 24 h with a phosphate buffer solution in order to remove unreacted BDDE.

(89) After manual homogenization for 30 min, a gel Y was obtained.

(90) c) Comparison of the Production Processes of Gel 2 and Gel Y

(91) As in Example 7, the gels 2 and Y were filled into glass syringes of 1 ml and extruded through a needle of 27G½. It was found that the two gels were easily injectable through the needle and the same comments as those set out in relation to Example 7 apply: the gel 2 according to the invention was found to be more homogeneous since its injection through the needle was more regular as that of gel Y. Surprisingly, although the gel 2 of the invention, which was not subjected to any grinding step into particles in order to facilitate its injectability, the complex “multistructured” structure of gel 2 exhibits a low ejection force similar to that of gel Y, which was subjected to a step of converting its strongly crosslinked phase into particles.

(92) As in Example 7, toluidine blue staining was performed on gel samples taken during the process of preparing gel 2 and gel Y.

(93) The following color tests were performed: For gel 2: for the partly crosslinked phases C and D (see FIGS. 1 and 2) and for the crosslinked product obtained from the partly crosslinked phases C and D (see FIG. 3), after neutralization to a neutral pH for three samples; For gel Y: for the particles Yp (see FIG. 6) and for the crosslinked product obtained by co-crosslinking of particles Yp (see FIG. 7), after neutralization to a neutral pH for two samples.

(94) The conclusion is the same as that of Example 7. In contrast to FIG. 6 (and also FIG. 7), in which NaHA crosslinked particles are clearly visible, the gel structure of FIGS. 1 to 3 relating to gel 2 according to the invention is not particulate.

(95) Therefore, the present invention allows one to obtain an easily injectable hydrogel, despite the fact that it was not grinded (partly or entirely) to form particles during its crosslinking process. Thus, the absence of co-crosslinked particles in the gel enables to achieve a significant improvement (compared to the gel obtained in EP 2 011 816): the safety profile in the short and long term is improved, in particular there is no migration of highly crosslinked particles in the long term as in EP 2 011 816; the bio-integration of the gel into tissues is enhanced (more homogeneous distribution) due to the absence of co-crosslinked particles.

(96) Finally, as in Example 7, it is important to note that the manufacture of the gel according to the invention has an economic advantage and minimizes the risk of particulate and bacterial contamination since the manufacturing process is significantly faster than that described in EP 2 011 816 (approximately 55 h for the gel according to the invention as compared to 83 h for the gel according to EP 2 011 816), and it does not require one or several complex and tedious milling steps as those required in EP 2 011 816.